1,4:3,6-dianhydro-D-glucitol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

LAB 3085

IUPAC Name:

LAB 3085

Details on test material:

Test substance : LAB 3085
Batch number : HW 11/10/00
I.P.L. Registration number : 010109
Physical form : white cristalline powder
Purity : 99.5% (molar purity)
Storage : at +4°C, protected from light and moisture
Solvent used : distilled water for injectable preparations, Fresenius #4807

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

The two strains TA 1535 and TA 100 show a base pair substitution in the hiis G gene (G-C in place of A-T). These two strains can be used to detect products which cause reverse base pair substitution.
The strain TA 1537 has, in the his C gene, a loop of several cytosines which give rise to a frameshift mutation. The strain TA 98 contains, in the his D gene, a sequence of four base pairs of cytosine-guanine (CG) which also give rise to a frameshift mutation.
The four strains also carry two other types of mutation : they are deficient in DNA repair mechanism (uvr B-) and show a change in the structure of cell-wall lipopolysaccharides (LPS) (rfa-). These two mutations make the bacteria more sensitive to genotoxic agents and more permeable to cell penetration by large molecules.
The R factor (present in strains TA 98 and TA 100), which is a pKM 101 ampicillin resistance plasmid, increases sensitivity to mutagenic agents via error-prone repair mechanisms (Maron and Ames, 1983).

Species / strain 2

Species / strain / cell type:

E. coli, other: WP2 pKM101

Details on mammalian cell type (if applicable):

Bacterial strains obtained from CROFTON-SLEIGH C, kept in the laboratory and preserved in liquid nitrogen :
WP2 pKM101:trp- presence of plasmid (R factor: pKM 101 ampicilline-resistant plasmid)
This Escherichia coli strains show the same type of mutation in the trp gene: the TAA ochre non-sens mutation. Moreover, they are deficient in DNA excision repair (uvrA-). That mutation makes the bacteria more sensitive to genotoxic agents such as UV which causes lesions in DNA.
The R factor increases sensitivity to mutagenic agents via error-prone repair mechanisms.

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

Bacterial strains obtained from CROFTON-SLEIGH C, kept in the laboratory and preserved in liquid nitrogen :
WP2 uvrA.pKM101: trp- ; uvrA- mutation presence of plasmid pKM 101 (R factor : pKM 101 ampicilline- resistant plasmid)
This Escherichia coli strains show the same type of mutation in the trp gene: the TAA ochre non-sens mutation. Moreover, they are deficient in DNA excision repair (uvrA-). That mutation makes the bacteria more sensitive to genotoxic agents such as UV which causes lesions in DNA.
The R factor increases sensitivity to mutagenic agents via error-prone repair mechanisms.

Metabolic activation:

with and without

Metabolic activation system:

hepatic microsomes from rat livers induced by Aroclor 1254- Incubation period 48 hours

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Controls 1

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for TA1535 and TA100

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for TA 1537

Controls 3

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for TA 98

Controls 4

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation for WP2pKM101

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-athramine

Remarks:

with metabolic activation for TA 1535, TA1537, TA98 and TA100

Controls 6

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation for WP2pKM101 and WP2 uvrA pKM101

Controls 7

Positive controls:

yes

Positive control substance:

other: Potassium dichromate

Remarks:

without metabolic activation for WP2 uvrA pKM101

Details on test system and experimental conditions:

After 48 hours of incubation at 37°C, the prototrophic mutant colonies that have developed in the plates are counted.

Evaluation criteria:

The results are expressed as the mean of mutants per plate and, for each concentration of the test product.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Species / strain:

bacteria, other: E. coli WP2 pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test compound LAB 3085 provided by ROQUETTE induced no mutagenic activity in the four Salmonella typhimurium and the two Escherichia coli strains tested.

Executive summary:

MUTAGENICITY TEST ON BACTERIA

(Salmonella typhimurium his- and Escherichia coli trp-)

USING B.N.AMES'S TECHNIQUE

This study was carried out in compliance with good laboratory practice.

Methods

Strains used: S.typhimurium: TA 1535, TA 1537, TA 98, TA 100

E.coli: WP2(pkM101) and WP2uvrA(pKM101)

Preliminary test of toxic activity: Carried out on 6 strains - Incubation period: 48 hours

Sterility test: Normal

Mutagenicity test: Carried out both with and without metabolic activation using hepatic microsomes from rat livers induced by Aroclor 1254 -Incubation period: 48 hours

Number of assays: 2 (the second assay with metabolic activation was performed according to pre-incubation method)

Initial solution: Distilled water

Limiting factor for maximum dose: Maximum dose according to OECD procedures

Doses used in main test: expressed as µg/plate

Without S9 mix

Strain	TA1535		TA1537		TA98		TA100		WP2(pKM101)		WP2uvrA(pKM101)
Assay	1	2	1	2	1	2	1	2	1	2	1	2
Doses µg/plate	0	0	0	0	0	0	0	0	0	0	0	0
	50	50	50	50	50	50	50	50	50	50	50	50
	150	150	150	150	150	150	150	150	150	150	150	150
	500	500	500	500	500	500	500	500	500	500	500	500
	1500	1500	1500	1500	1500	1500	1500	1500	1500	1500	1500	1500
	5000	5000	5000	5000	5000	5000	5000	5000	5000	5000	5000	5000

With S9 mix

Strain	TA1535		TA1537		TA98		TA100		WP2(pKM101)		WP2uvrA(pKM101)
Assay	1	2 *	1	2*	1	2*	1	2*	1	2*	1	2 *
Doses µg/plate	0	0	0	0	0	0	0	0	0	0	0	0
	50	50	50	50	50	50	50	50	50	50	50	50
	150	150	150	150	150	150	150	150	150	150	150	150
	500	500	500	500	500	500	500	500	500	500	500	500
	1500	1500	1500	1500	1500	1500	1500	1500	1500	1500	1500	1500
	5000	5000	5000	5000	5000	5000	5000	5000	5000	5000	5000	5000

* assay with pre-incubation

Results:

The test compound LAB 3085 provided by ROQUETTE induced no mutagenic activity in the four Salmonella typhimurium and the two Escherichia coli strains tested.